DNA topoisomerases catalyze changes in DNA topology through a concerted mechanism of DNA strand breakage and rejoining. The transient cleavage of the DNA backbone is accompanied by the formation of a covalent enzyme- DNA intermediate. These intermediates, termed cleavable complexes, are reversibly stabilized by a number of therapeutically important drugs, including camptothecin, a potent neoplastic agent, that specifically targets eukaryotic DNA topoisomerase I. This enzyme plays an important role in DNA replication, RNA transcription and DNA recombination and is highly conserved among eukaryotes. This is reflected in similarities in enzyme structure, function and sensitivity to camptothecin. The cytotoxic activity of camptothecin is S-phase specific, presumably resulting from the interaction of DNA replication forks with the drug- stabilized enzyme-DNA cleavable complex. However, the exact nature of the molecular interactions required for the formation of these cleavable complexes and their subsequent conversion into lethal events remains unknown. This application proposes to define the specific molecular interactions required for camptothecin-induced cell lethality in the yeast Saccharomyces cerevisiae. Earlier reports of the restoration of camptothecin sensitivity to yeast cells expressing either the yeast or human DNA topoisomerase I gene, suggest that the processes involved in converting the drug stabilized complex into a lethal lesion could be experimentally addressed in this genetically tractable system. specifically, the particular enzyme domains and amino acid residues required for a productive interaction with camptothecin and DNA will be defined, as will the effects of camptothecin on enzyme structure and DNA binding. Two classes of DNA topoisomerase I mutants, one exhibiting resistance to camptothecin and the other exhibiting a camptothecin- independent lethal phenotype, will similarly be characterized. In addition, a genetic screen will be used to identify second site suppressors of camptothecin sensitivity. Subsequent identification of these genes and their in vivo function(s) will elucidate the molecular interactions required for drug-induced cell death. These studies, as well as those involving second site suppressors of camptothecin- independent lethal mutants, will not only further our understanding of the mechanism of drug-induced lethality, but will also lead to greater understanding of how normal cellular functions can be perturbed to cause cell death. As camptothecin analogues are currently being developed for clinical application to cancers including ovarian and lung, these results will have much broader application in the design and development of new therapeutics.